ATP对透析乌贼轴突中单价阳离子与钠泵之间相互作用的影响。

The effects of ATP on the interactions between monovalent cations and the sodium pump in dialysed squid axons.

作者信息

Beaugé L, Di Polo R

出版信息

J Physiol. 1981 May;314:457-80. doi: 10.1113/jphysiol.1981.sp013719.

DOI:10.1113/jphysiol.1981.sp013719

PMID:6273535

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1249445/

Abstract

The efflux of Na in dialysed axons of the squid has been used to monitor the sidedness of the interactions of the Na pump with Na(+) ions, K(+) ions and ATP. The axons were under conditions such that most of the Na efflux went through the Na pump by means of a complete cycle of ATP hydrolysis.2. With 310 mm-K(i) (+), 70 mm-Na(i) (+) and 10 mm-K(+) artificial sea water (ASW) more than 97% of the Na efflux was abolished by removal of ATP. The efflux of Na was stimulated by ATP with a K((1/2)) of about 200 mum. This is similar to the K((1/2)) of 150 mum found for the ATP dependence of a ouabain-sensitive Na,K-ATPase activity in membrane fragments isolated from squid optical nerves.3. A 100-fold reduction in the ATP concentration (from 3-5 mm to 30-50 mum) increased the apparent affinity of the Na pump for K(o) (+) about 8-fold. In addition, the maximal rate of K(o) (+)-stimulated Na efflux was reduced by a similar factor. Analogous results were seen in axons dialysed with 310 mm-K(i) (+) or without K(i) (+).4. The relative effectiveness of external monovalent cations as activators of the Na efflux was a function of the ATP concentration inside the axon. With 3-5 mm-ATP the order of effectiveness was K(+) > NH(4) (+) > Rb(+). With 30-50 mum-ATP the sequence was NH(4) (+) >> K(+) >> Rb(+). These results were not affected by the removal of K(i) (+).5. When the ATP concentration was 3 mm and the Na(i) (+) concentration 70 mm, the removal of K(i) (+) produced a slight and reversible increase in the total efflux of Na (15%) and no change in the ATP-dependent Na efflux. When the ATP concentration was reduced to 30-50 mum, or the Na(i) (+) concentration lowered to 5-10 mm, the removal of K(i) (+) reversibly increased the total and the ATP-dependent efflux of Na. The largest increase in Na efflux was seen when both ATP and Na(i) (+) were simultaneously reduced. The ATP-dependent extra Na efflux resulting from the exclusion of K(i) (+) was abolished by 10(-4)m-ouabain in the sea waters.6. The increase in the ATP-dependent Na efflux observed in axons dialysed with 0 K(i) (+) + 10 mm-K(+) ASW was not seen in axons perfused with 310 mm-K(i) (+) + 450 mm-K(+) ASW. However, both experimental conditions gave rise to a similar (and small) ATP-independent and ouabain-insensitive efflux of Na. This indicates that the effects on the Na pump of removing K(i) (+) are not due to the simultaneous membrane depolarization. In addition, it suggests that K(i) (+) has an inhibitory effect on the Na pump, and that that effect is antagonized by Na(i) (+) and ATP.7. The present results are consistent with the idea that the same conformation of the Na pump (and Na,K-ATPase) can be reached by interaction with external K(+) after phosphorylation and with internal K(+) before rephosphorylation. This enzyme conformation produces an enzyme-K complex from which K(+) ions are not easily released unless high concentrations of ATP are present. This also stresses a non-phosphorylating regulatory role of ATP.

摘要

乌贼透析轴突中Na⁺的外流已被用于监测钠泵与Na⁺离子、K⁺离子和ATP相互作用的方向性。轴突处于这样的条件下，即大部分Na⁺外流通过ATP水解的完整循环经钠泵进行。
在含有310 mM - K⁺（内）、70 mM - Na⁺（内）和10 mM - K⁺的人工海水（ASW）中，去除ATP可使超过97%的Na⁺外流被消除。ATP以约200 μM的K₁/₂刺激Na⁺外流。这与从乌贼视神经分离的膜片段中哇巴因敏感的Na,K - ATP酶活性对ATP依赖性所发现的150 μM的K₁/₂相似。
ATP浓度降低100倍（从3 - 5 mM降至30 - 50 μM）使钠泵对K⁺（外）的表观亲和力增加约8倍。此外，K⁺（外）刺激的Na⁺外流的最大速率降低了类似的倍数。在用310 mM - K⁺（内）透析或无K⁺（内）的轴突中也观察到类似结果。
外部单价阳离子作为Na⁺外流激活剂的相对有效性是轴突内ATP浓度的函数。在3 - 5 mM - ATP时，有效性顺序为K⁺＞NH₄⁺＞Rb⁺。在30 - 50 μM - ATP时，顺序为NH₄⁺＞＞K⁺＞＞Rb⁺。这些结果不受去除K⁺（内）的影响。
当ATP浓度为3 mM且Na⁺（内）浓度为70 mM时，去除K⁺（内）使Na⁺的总外流轻微且可逆地增加（15%），而ATP依赖性Na⁺外流无变化。当ATP浓度降至30 - 50 μM或Na⁺（内）浓度降至5 - 10 mM时，去除K⁺（内）可逆地增加Na⁺的总外流和ATP依赖性外流。当ATP和Na⁺（内）同时降低时，Na⁺外流增加最大。海水中10⁻⁴M哇巴因可消除因排除K⁺（内）导致的ATP依赖性额外Na⁺外流。
在用0 K⁺（内） + 10 mM - K⁺ ASW透析的轴突中观察到的ATP依赖性Na⁺外流增加，在用310 mM - K⁺（内） + 450 mM - K⁺ ASW灌注的轴突中未见到。然而，两种实验条件都产生了类似的（且小的）不依赖ATP和哇巴因不敏感的Na⁺外流。这表明去除K⁺（内）对钠泵的影响不是由于同时发生的膜去极化。此外，这表明K⁺（内）对钠泵有抑制作用，且该作用被Na⁺（内）和ATP拮抗。
目前的结果与以下观点一致：钠泵（和Na,K - ATP酶）的相同构象可通过磷酸化后与外部K⁺相互作用以及再磷酸化前与内部K⁺相互作用而达到。这种酶构象产生一种酶 - K复合物，除非存在高浓度的ATP，K⁺离子不易从该复合物中释放。这也强调了ATP的非磷酸化调节作用。